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United States Patent |
5,049,666
|
Lin
,   et al.
|
September 17, 1991
|
Water soluble TMC-crown formazans for detecting lithium
Abstract
Novel compounds and assay methods are provided for determining the presence
of lithium in serum, plasma, urine or other sample without
deproteinization. The novel compounds are water soluble derivatives of
TWC-crownformazans and provide signal enhancement by increased absorbance
of the dye-lithium complex over the dye anion.
Inventors:
|
Lin; Cheng-I (San Jose, CA);
Pirio; Marcel (San Jose, CA)
|
Assignee:
|
Syntex (U.S.A.) Inc. (Palo Alto, CA)
|
Appl. No.:
|
164025 |
Filed:
|
March 4, 1988 |
Current U.S. Class: |
540/469; 534/652; 564/305; 568/644 |
Intern'l Class: |
D06P 050/04 |
Field of Search: |
540/469
534/652
564/305
568/644
|
References Cited
U.S. Patent Documents
4370145 | Jan., 1983 | Schwaiger et al. | 8/549.
|
4843158 | Jun., 1989 | Smith | 540/469.
|
4892937 | Jan., 1990 | Delton et al. | 534/652.
|
Other References
Sitnikova, R. V. et al., Chem. Abstr. 97 174,017d.
Dziomko, V. M. et al., Chem. Abstr. 96 52,283w.
Ostrovskaya, Y. M. et al., Chem. Abstr. 100:95775b.
Chemical Abstracts, 1982-1986 Chemical Substance Index, p 23329CS.
|
Primary Examiner: Shah; Mukund J.
Assistant Examiner: Grumbling; Matthew V.
Attorney, Agent or Firm: Leitereg; Theodore J., Precivale; Shelley G.
Parent Case Text
This is a division of Ser. No. 06/866,821 filed May 23, 1986, now issued as
U.S. Pat. No. 4,742,010, incorporated herein by reference.
Claims
What is claimed is:
1. A compound of the formula:
##STR5##
wherein: .alpha. and .beta. are taken together to form
##STR6##
wherein: R.sub.1 is an electron withdrawing group selected from the group
consisting of --NO.sub.2, --SO.sub.2 CF.sub.3, CONX.sub.2, --SO.sub.2
NX.sub.2 and --CN, and X is independently selected from the group
consisting of hydrogen, methyl and ethyl;
R.sub.2 and R.sub.5 are independently selected from the group consisting of
hydrogen, methoxy, carboxymethoxy, nitro, chlorine, fluorine and bromine;
R.sub.3 and R.sub.4 are independently selected from the group consisting of
hydrogen and a hydrophilic substituent consisting of from one to twelve
atoms other than hydrogen, which atoms are selected from the group
consisting of carbon, oxygen, nitrogen, phosphorous, sulfur and halogen;
with the proviso that not more than three of R.sub.3 and R.sub.4 are
hydrogen.
2. The salt of a compound according to claim 1 wherein .alpha. and .beta.
are taken together to form
##STR7##
resulting from replacement of the hydrogen on the nitrogen atom with a
metal selected from the group consisting of lithium, sodium and potassium.
3. A lithium salt according to claim 2.
4. A compound according to claim 1 wherein at least one of R.sub.3 and
R.sub.4 is independently selected from the group consisting of amines,
amides, ethers, carboxylic, sulfonic, phosphoric and phosphonic acids and
esters thereof and alcohols other than phenol.
5. A compound according to claim 1 wherein at least one of R.sub.2 and
R.sub.5 is hydrogen.
6. A compound according to claim 4 wherein R.sub.3 is hydrogen or
--SO.sub.3 H.
7. A compound according to claim 4 wherein R.sub.4 is selected from the
group consisting of --CO.sub.2 NH.sub.2, --SO.sub.3 H, CO.sub.2 H and
--CONHCH.sub.2 COOH.
8. A compound of the formula:
##STR8##
wherein: R.sub.1 is an electron withdrawing group, selected from the group
consisting of --NO.sub.2, --SO.sub.2 CF.sub.3, CONX.sub.2, --SO.sub.2
NX.sub.2 and --cn, and X is independently selected from the group
consisting of hydrogen, methyl and ethyl;
R.sub.2 and R.sub.5 are independently selected from the group consisting of
hydrogen, methoxy, carboxymethoxy, nitro, chlorine, bromine and fluorine;
and
R.sub.3 and R.sub.4 are independently selected from the group consisting of
hydrogen and a hydrophilic substituent consisting of from one to twelve
atoms other than hydrogen, which atoms are selected from the group
consisting of carbon, oxygen, nitrogen, phosphorous, sulfur and halogen;
with the proviso that not more than three of R.sub.3 and R.sub.4 are
hydrogen.
9. The salt of compound according to claim 8 wherein .alpha. and .beta. are
taken together to form
##STR9##
resulting from replacement of the hydrogen on the nitrogen atom with a
metal selected from the group consisting of lithium, sodium and potassium.
10. A lithium salt according to claim 9.
11. A compound according to claim 8 wherein at least one of R.sub.3 and
R.sub.4 is independently selected from the group consisting of amines,
amides, ethers, carboxylic, sulfonic, phosphoric and phosphonic acids and
esters thereof and alcohols other than phenol.
12. A compound according to claim 8 wherein at least one of R.sub.2 and
R.sub.5 is hydrogen.
13. A compound according to claim 11 wherein R.sub.3 is hydrogen or
SO.sub.3 H.
14. A compound according to claim 11 wherein R.sub.4 is selected from the
group consisting of --CO.sub.2 NH.sub.2, --SO.sub.3 H, --CO.sub.2 H, and
--CONHCH.sub.2 COOH.
15. A compound of the formula:
##STR10##
wherein: R.sub.1 is NO.sub.2 ;
R.sub.2, R.sub.3 and R.sub.5 are H; and
R.sub.4 is CO.sub.2 H.
16. A compound of the formula:
##STR11##
wherein: R.sub.1 is SO.sub.2 CF.sub.3 ;
R.sub.2, R.sub.3 and R.sub.5 are H; and
R.sub.4 is CO.sub.2 H.
17. A compound of the formula:
##STR12##
wherein: R.sub.1 is NO.sub.2 ;
R.sub.2, R.sub.3 and R.sub.5 are H; and
R.sub.4 is CONHCH.sub.2 CO.sub.2 H.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to new methods and compounds for determining lithium
level in serum or other samples suspected of containing lithium, and more
particularly to a homogeneous colorimetric method that is performed
directly on the sample without deproteinization. Lithium in the form of
lithium carbonate is administered to manic-depressive patients. The
therapeutic range of lithium ion in plasma is quite narrow, namely, 0.8 to
1.2 mM. It is important to monitor the lithium level in such patients
because of the toxic side effects that appear when the lithium level in
blood exceeds the recommended level.
Current methods for detecting and determining lithium generally involve
flame emission or atomic absorption methods. These techniques are
disadvantageous because they are slow and labor intensive.
2. Description of the Related Art
Recently, colorimetric tests have been suggested for lithium analysis. R.
V. Sitnikova et al in Zh. Anal. Khim., 37(4) 611-13(1982) disclose use of
TMC-crownformazan (15, 16-dihydro-7-cyano-5H, 17H-dibenzo [b,i]
[1,11,4,5,7,8]-dioxa-tetraazacyclotetradecine) as a reagent to complex
lithium. A spectometric method for determining lithium in plasma and urine
was suggested using either the aforementioned crownformazan or a
pyrazolone by R. V. Sitnikova et al in Lab. Delo. (3) 142-5(1980).
Use of TMC-crownformazan in lithium assays is disadvantageous. Such tests
require the use of nearly water-free organic solvents, and therefore
require that the sample be pretreated to remove protein which otherwise
precipitates in the organic solvents and interferes with the assay.
Further, only slight chromophoric changes are observed when crownformazan
is used to complex lithium ion. Pyrazolones have been found to be both
less selective and less sensitive than TMC-crownformazan. Further, tests
using pyrazolones require deproteinization.
Use of an arsenophenyl ligand called Thoron in an alkaline acetone medium
has been suggested by J. K. Trautman et al in Talanta 30(8) 587-91 (1983)
for spectrophotometric determination of lithium. The dye solution
suggested by Trautman et al is disadvantageous because it has a short
shelf life, and sodium and other ions interfere with the results.
Additionally, lithium assays using Thoron require deproteinization.
SUMMARY OF THE INVENTION
Methods and compositions are provided for the determination of the presence
of lithium in a sample suspected of containing lithium. The method is a
reliable and easily performed chromogenic assay for lithium in a sample
suspected of containing lithium, including serum, plasma, urine, or the
like without deproteinization. The method is carried out using the novel
crownformazan chelating agents of the present invention. Signal
enhancement is due to the enhanced absorbance of the dye-lithium complex
over the dye anion. The compositions of the present invention are novel
solubilized derivatives of TMC-crownformazan.
In the method of the present invention the novel compounds are combined in
an aqueous assay medium with a sample suspected of containing lithium, and
the absorbance of the medium is measured.
Combinations of reagents are provided as kits to enhance the observed
sensitivity of the assay by providing for ratios to substantially optimize
the sensitivity for the range of interest of the lithium analyte. In
addition to an aqueous solution of the TMC-crownformazan compounds of the
present invention, the test kits can include a cosolvent, for example,
DMSO, or DMF.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
The present invention relates to a method for assaying for lithium. The
method entails combining in an aqueous medium a test sample suspected of
containing lithium ion with a novel crownformazan of the present invention
as a complexing agent or chomogenic ionophore.
The aqueous medium is selected to avoid precipitation of components in the
sample. It was found that the need for deproteinization of a sample in
assays for lithium could be avoided by using novel water soluble
derivatives of TMC-crownformazans in an aqueous assay medium. This medium
can additionally contain polar cosolvents at concentrations below those
that cause precipitation of serum proteins. The cosolvents increase the
lithium affinity, and thus the sensitivity, of the assay. These cosolvents
are usually polar organic substances of 1 to 12 atoms other than hydrogen,
selected from carbon, oxygen, nitrogen, phosphorous, sulfur and halogen.
Suitable cosolvents include dimethylsulfone, dimethylacetamide,
tetrahydrofuran, dimethylsulfoxide (DMSO), sulfolane trimethylphosphate,
dimethylformamide (DMF), dioxane, and the like. Within limits, the greater
the amount of cosolvent used, the greater the sensitivity of the assay.
The main limitation regarding the choice and concentration of the
cosolvent is that sample components must not be precipitated. If the
components are precipitated, a turbid solution results that will prevent
accurate spectroscopic measurements. Therefore, cosolvent concentrations
generally range from 0-75%, more usually 30-60%. Generally, cosolvent
concentration depends on the nature of the sample, and the concentration
of the lithium to be determined. All solutions are preferably filtered
free of particulate matter to avoid light scattering.
Primarily, the method of the present invention is employed for
determinations of lithium in blood and blood fractions such as serum and
plasma; however, the method is usuable for determinations of lithium in
other body fluids such as urine, saliva, and the like. In general, the
method of the present invention offers significant advantages over
alternative methods when aqueous lithium solutions are to be analyzed. The
present method offers particularly significant advantages when the aqueous
lithium solution contains solutes that would precipitate in organic
solvents.
The method of the present invention is usually conducted by combining a
measured amount of an aqueous sample suspected of containing lithium or
containing an inknown concentration of lithium with a reagent comprised of
a novel crownformazan and an amine or ammonium hydroxide or an alkali
metal hydroxide, other than lithium hydroxide. The aqueous solution is
sufficiently alkaline so as to cause at least partial ionization of the
crownformazan in the absence of lithium ion. Generally the ionization is
about 5-100%. Preferably, ionization should be at least 80%. Potassium
hydroxide in an aqueous solvent is preferred, but sodium hydroxide, or
cesium or rubidium hydroxides and the like, are also usable. If the sample
is believed to contain a high concentration of metal cation, for example
sodium ion, it will usually be desirable to include sufficient
concentration of that ion in the reagent so that the chromophoric response
due to the ion is nearly saturated, and therefore will not change
significantly as a result of introducing the ion from the sample into the
assay mixture. If the sample is likely to contain a high concentration of
sodium ion, for example, if a serum or urine is used, then an excess of
sodium ion is added to the medium to minimize signal fluctuations due to
serum sodium variations in the serum or urine sample. For this purpose,
sodium ion generally is included in the reagent such that the sodium ion
concentration is in excess of that expected after addition of the serum
sample. If the sample is not believed to have a high sodium concentration,
excess sodium ion is generally not added.
The method of the present invention is generally carried out at a pH in the
range of 8 to 14, and preferably in the range of 10 to 14. Various bases
may be used to achieve the desired pH during the determination.
Illustrative bases include alkyl almines, Tris buffer, potassium
phosphate, ammonia, guanidine and the like. The particular base employed
is not critical to this invention, but in individual assays one base may
be preferred over another.
Moderate temperatures are normally used for carrying out the method of the
present invention, and usually constant temperatures during the period for
conducting the method. The temperatures for the determination will
generally range from about 0.degree. to 50.degree. C., more usually from
about 15.degree. to 40.degree. C.
Considerations such as whether the assay is qualitative, semi-quantitative
or quantitative will determine the concentration of the complexing agent.
Generally, the concentration range of the lithium ion will determine the
concentration of the complexing agent; however, the final concentration of
the complexing agent is preferably determined empirically to optimize the
sensitivity of the assay over the range of interest.
The lowest useful concentration of crownformazan is determined by the
ability to detect the absorbance change and is thus dependent on various
factors including the spectrometer sensitivity, path length of the light
beam through the solution and the required range of the assay. The
concentration of the crownformazan should be at least as great as the
highest lithium concentration that is to be detected. In general, the
lowest concentration of crownformazan will be about fivefold less than the
dissociation constant of the lithium-crownformazan chelate under the assay
conditions.
It will be understood that the highest useful concentration of
crownformazan is also dependent in part on the spectrometric measurement
capability of the instrument. Sufficient light must be transmitted to
permit measurement of the difference between the absorbance with varying
concentrations of lithium. In general, as the concentration of the
crownformazan increases, the sensitivity of the assay will decrease
because the ratio of modulated to unmodulated signal will decrease with
increasing crownformazan concentration. Thus, the highest concentration of
crownformazan will usually be no greater than 50 times, and preferably 20
times the lowest concentration of lithium that is to be detected.
The concentration of lithium in the sample to be assayed will generally
vary from 0.1-1000 , mM usually about 0.1-2 mM, more usually from about
0.25 to 1.5 mM.
To determine the concentration of lithium, the assay response should be
related to the response produced by a standard or calibrator containing a
known concentration of lithium. Usually a standard curve will be
constructed using two or more such calibrators. When a sufficiently high
concentration of crownformazan is used, so that it never becomes more than
about 40% bound by lithium even at the highest lithium concentration, the
standard curve will approach linearity. Under these conditions, only one
or two calibrators may be required.
A spectroscopic measurement is made at a wavelength that is defined for
each novel crownformazan. Preferably, no separation or centrifugation
steps are included in the assay. The measurement wavelength will usually
be near the wavelength at which the absorbance of the crownformazan varies
maximally as a function of lithium concentration. The wavelengths are
generally in the range of about 480 to 600 nm, and more usually in the
range of 480 to 520 nm.
Spectroscopic measurements may be direct or may be by difference. When by
difference, an assay solution is placed in the reference compartment that
is nearly identical to the assay solution containing the sample except
that the assay solution in the reference will contain a known amount of
lithium or no lithium.
The novel crownformazan chelating agents of the present invention form
complexes with lithium. The agents are used without deproteinization of a
sample to be analyzed in a colorimetric assay system that is capable of
measuring pharmacologically significant levels of lithium with great
accuracy and with substantially no interference from similar elements,
such as sodium and potassium, and the like.
Preferably, solubilizing groups are attached para to the nitrogens on the
aromatic rings (R.sub.4) of the crownformazans of the present invention.
Substituents capable of rendering the composition soluble comprise a group
of from 1 to 12 atoms other than hydrogen, selected from carbon, oxygen,
nitrogen, phosphorous, sulfur and halogen. Solubilizing groups include
carboxylic, phosphonic, sulfonic and phosphoric acids, amines, ethers,
amides and alcohols other than phenols.
In such a case, the preferred solubilizing groups are those containing
COOH, CONH.sub.2, and CONHCH.sub.2 COOH. Independently selected
solubilizing agents may also be attached meta to the nitrogens on the
aromatic rings (R.sub.3) of the crownformazan. In such a case, the
preferred solubilizing group is one containing a sulfonic acid.
Additionally, the crownformazan of the present invention should contain an
electron withdrawing group, such as, for example, CN, NO.sub.2, SO.sub.2
NX.sub.2, CONX.sub.2 and SO.sub.2 CF.sub.3 where X is independently
selected from a group consisting of hydrogen and lower alkyl of about 1 to
2 carbon atoms, more preferably 1 carbon atom. NO.sub.2 is the preferred
electron withdrawing substituent. The electron withdrawing group is
attached to the carbon atom of the nitrogen containing bridge of the
crownformazan.
The novel compounds of the present invention generally have the following
structure:
##STR1##
wherein:
.alpha. and .beta. are NO.sub.2 or NH.sub.2 or can be taken together to
form
##STR2##
R.sub.1 is an electron withdrawing group, such as, for example, --NO.sub.2,
--SO.sub.2 CF.sub.3, --CONX.sub.2, SO.sub.2 NX.sub.2, or CN, wherein X is
independently selected from a group consisting of hydrogen or lower alkyl
containing about 1 to 2 carbon atoms, and more preferably 1 carbon atom.
R.sub.2 and R.sub.5 are independently selected from the group consisting of
hydrogen, methoxy, carboxymethoxy, nitro, chlorine, fluorine and bromine,
preferably hydrogen and fluorine.
R.sub.3 and R.sub.4 are independently selected from a group consisting of
hydrogen and a hydrophilic substituent consisting of from 1 to 12 atoms
other than hydrogen, which atoms are selected from the group consisting of
carbon, oxygen, nitrogen, phosphorous, sulfur and halogen; with the
proviso that no more than three of R.sub.3 and R.sub.4 are hydrogen.
Preferably at least one of R.sub.3 and R.sub.4 is selected from the group
consisting of amines, amides, ethers, and carboxylic, phosphonic, sulfonic
and phosphoric acids, esters thereof and alcohols other than phenol.
R.sub.3 is preferably hydrogen or --SO.sub.3 H. R.sub.4 is preferably
hydrogen, CO.sub.2 NH.sub.2, --CO.sub.2 H, --SO.sub.3 H, or --CONHCH.sub.2
COOH.
A preferred embodiment has the aforementioned structure wherein .alpha. and
.beta. are both NH.sub.2 and R.sub.3 is --SO.sub.3 H. In an additional
preferred embodiment .alpha. and .beta. are both --NO.sub.2 and R.sub.4 is
--CO.sub.2 H.
In another preferred embodiment the novel complexing agents of the present
invention have the following structure:
##STR3##
wherein:
R.sub.1 is an electron withdrawing group, such as, for example, --NO.sub.2,
--SO.sub.2 CF.sub.3, --CONX.sub.2, SO.sub.2 NH.sub.2, or CN, wherein X is
independently selected from a group consisting of hydrogen or a lower
alkyl containing about 1 to 2 carbon atoms, and more preferably 1 carbon
atom.
R.sub.2 and R.sub.5 are independently selected from the group consisting of
hydrogen, methoxy, carboxymethoxy, chlorine, bromine or fluorine, and
preferably hydrogen and fluorine.
R.sub.3 and R.sub.4 are independently selected from a group consisting of
hydrogen and a hydrophilic substituent consisting of from 1 to 12 atoms
other than hydrogen. The atoms are selected from the group consisting of
carbon, oxygen, nitrogen, phosphorous, sulfur and halogen; with the
proviso that not more than three of R.sub.3 and R.sub.4 are hydrogen.
Preferably, at least one of R.sub.3 and R.sub.4 is selected from the group
consisting of amines, amides, ethers, carboxylic, phosphonic, sulfonic and
phosphoric acids, and alcohols other than phenol. R.sub.3 is preferably
hydrogen or --SO.sub.3 H. R.sub.4 is preferably hydrogen, CO.sub.2
NH.sub.2, --CO.sub.2 H, --SO.sub.3 H, or --CONHCH.sub.2 COOH.
In a further preferred embodiment the novel complexing agents have the
following structure:
##STR4##
wherein: R.sub.1 is NO.sub.2 ; R.sub.2, R.sub.3 and R.sub.5 are hydrogen;
and R.sub.4 is CO.sub.2 H.
In another preferred embodiment the novel complexing agents have the
aforementioned structure wherein: R.sub.1 is SO.sub.2 CF.sub.3 ; R.sub.2,
R.sub.3 and R.sub.5 are hydrogen; and R.sub.4 is CO.sub.2 H.
In an additional preferred embodiment the novel complexing agents have the
aforementioned structure wherein: R.sub.1 is NO.sub.2 ; R.sub.2, R.sub.3
and R.sub.5 are hydrogen; and R.sub.4 is CONHCH.sub.2 CO.sub.2 H.
As a matter of convenience, the reagents for conducting an assay according
to the present invention, can be provided in a kit in a packaged
combination in predetermined amounts for use in assaying for lithium. The
kit can comprise an aqueous alkaline solution, preferably potassium
hydroxide, and a solution of the novel crownformazan composition of the
present invention, for example, the nitro-bis carboxyl derivatives in an
aqueous solvent solution. It is, also desirable for the kit to include an
aqueous solution of about 40% to about 60% of a polar organic solvent,
preferably dimethylsulfoxide. The kit may contain optional additional
ingredients or ancillary agents as necessary such as, e.g., surfactants,
and antimicrobial agents.
EXPERIMENTAL
All temperatures are in centigrade. All parts are by weight, except liquids
which are by volume unless otherwise indicated. The following
abbreviations are used: DMSO-dimethylsulfoxide; THF-tetrahydrofuran;
NHS-N-hydroxy succinimide; EtOAc-ethylacetate; TLC-thin layer
chromatography.
EXAMPLE I
7-nitro-3,11-disulfo-16,17-dihydro-5H, 15H-dibenzo [b,i] [1,11,4,5,7,8]
dioxatetraazacyclotetradecin(V)
A 500-ml round-bottom flask was equipped with a CaCl.sub.2 drying tube and
a magnetic stir bar and was charged with 11.1 g (0.090 mols) of
nitrophenol, 8.24 g (0.040 mols) of 1,3-dibromopropane and 10 g (0.078
mols) of anhydrous K.sub.2 CO.sub.3. The mixture was stirred magnetically
and heated at 50.degree. C. for 8 hours, and then an additional 4 g (0.032
mols) of nitrophenol were added and heating was continued overnight. The
solvent was removed under vacuum with heating, and the residue was
dissolved with EtOAc. The organic phase was extracted twice with 100 ml
10% K.sub.2 CO.sub.3 solution. The organic extract was dried over
MgSO.sub.4 and concentrated in vacuo. The residue was crystallized from
EtOAc/hexane, and the product (II) was found to have a melting point of
109.degree.-110.degree. C.
2 g (0.0063 mols) of the product (II) were dissolved in 20 ml of fuming
sulfuric acid (20% H.sub.2 SO.sub.4.SO.sub.3) and the solution turned dark
brown. After all the solid had dissolved and was stirred for an additional
5 minutes, the reaction was quenched with 50 ml of ice. The pH was
adjusted to 5.0 with a saturated NaOH solution. After standing overnight
at room temperature, a crystalline precipitate formed. The solid was
filtered, and the cake was washed with ice cold water. The product (III)
weighted 1.5 g after being dried over P.sub.2 O.sub.5 in vacuo.
A 250-ml Parr hydrogenation vessel was charged with 500 mg (1.0 mmols) of
the product (III), 50 ml (6/4) water/methanol, and 100 mg 10% palladium on
carbon and, then was subjected to 50 psi hydrogen for 2.0 hr. The reaction
mixture was filtered and the filtrate was concentrated in vacuo at
60.degree. C. 400 mg of product IV was recovered.
300 mg (0.717 mmols) of product IV, 100 ml of water, and 2.0 ml
concentrated HCl were mixed in a 500 ml flask and the solution was cooled
in an ice bath to .sup..about. 5.degree. C., thereafter 0.148 g (0.002
mols) of NaNO.sub.2 in 5 ml of water were added dropwise. After 3.5 hr,
0.131 g (0.002 mols) of nitromethane were added. The solution was stirred
vigorously with dropwise addition of concentrated NH.sub.4 OH and a dark
cherry red solution formed. After the solution was stirred for one hour at
pH 7.5-8.0, the solution was made acidic (pH 6.0) with 10% HCl and then
concentrated to dryness at 40.degree. C. in vacuo. Half of the material
was subjected twice to chromatography on 5-20.times.20 cm 500 micron
Analtech avicel F cellulose plates, eluant (1/1) methanol-water. The
appropriate red band was extracted with water, concentrated in vacuo
yielding 125 mg of product, dried under vacuum at 100.degree. over P.sub.2
O.sub.5 and found to have a melting point greater than 300.degree. C.
EXAMPLE II
3,3'-(1,3-dioxopropyl)-4,4'-dinitrobismethyl Benzoate (VIII)
A 250-ml round-bottom flask equipped with a drying tube, condenser, and
magnetic stir bar was charged with 10.7 g (0.058 mols) of
3-hydroxy-4-nitrobenzoic acid (VI) and 100 ml of thionyl chloride and
refluxed overnight. The cooled reaction mixture was concentrated in vacuo
with heating, and the residue was dissolved with 150 ml of ice-cold
anhydrous methanol. After stirring at ambient temperature overnight, the
solution was concentrated in vacuo and the product was crystallized from
boiling hexane to give product VII having a melting point of
88.degree.-89.degree. C.
5 g (0.025 mols) of product VII were mixed with 6.9 g (0.05 mols) of
anhydrous K.sub.2 CO.sub.3 and 2.2 g (0.011 mols) of 1,3-dibromopropane in
a 500-ml round-bottom flask equipped with a drying tube and magnetic stir
bar. The reaction mixture was stirred at 50.degree. C. for 16 hrs. An
aliquot of the reaction mixture was spotted on TLC (CH.sub.2 Cl.sub.2
silica gel plate).
The cooled reaction mixture was vacuum filtered and the solid was washed
with DMF. The filtrate was added to 1.5 liters of ice-cold water and
extracted several times with 500 ml CH.sub.2 Cl.sub.2. The organic
extracts were back washed with several times 200 ml 1N NaOH. The CH.sub.2
Cl.sub.2 phase was dried with MgSO.sub.4 and concentrated in vacuo.
Product VIII was crystallized from acetone-ether and had a melting point
of 148.degree.-49.degree. C.
EXAMPLE III
3,3'-(1,3-dioxopropyl)-4,4'-diaminobismethyl benzoate (IX)
1 g (0.0023 mols) of product VIII was dissolved in 250 ml ethyl acetate in
a ml Parr hydrogenation flask with 200 mg 10% palladium on carbon and
hydrogenated under 50 psi of H.sub.2 for 15 minutes. The solution was
filtered and filtrate concentrated in vacuo. The product IX was
crystallized from ethyl-acetate-hexane and had a melting point of
151.degree. to 152.degree. C.
EXAMPLE IV
7-carbonitrile-2,12-dimethoxycarbonyl, 16,17-dihydro-5H, 15H-dibenzo [b,i]
[1,11,4,5,7,8] dioxatetraacyclotetradecin (X)
A mixture of 750 mg (2 mmols) of the product of Example III and 4.5 ml 1N
hydrochloric acid in 200 ml of water was cooled to 2.degree. C. 276 mg (4
mmols) of sodium nitrite in 10 ml of water were added dropwise to the
solution and after 3 hours the diazonium solution was added dropwise to a
stirring solution of 212 mg (2.5 mmols) of cyanoacetic acid and 1.5 ml 6N
NaOH in 100 ml water. The solution was stirred overnight at ambient
temperature and acidified with concentrated HCl. A dark red precipitate
was formed and the solid material was collected and then dissolved in
CH.sub.2 Cl.sub.2, dried with MgSO.sub.4 and concentrated in vacuo.
Chromatography purification on Analtech silica gel GF 1000 micron plates,
eluant ethyl acetate hexane. The product (X) was crystallized from
chloroform yielded dark brown crystals with a melting point of more than
300.degree. C.
EXAMPLE V
7-trifluoromethylsulfonyl-2,12-dimethoxycarbonyl-16,17-dihydro-5H,15H-diben
zo [b,i] [1,11,4,5,7,8] dioxatetraacyclotetradecin (XI)
250 mg (0.668 mmols) of the product of Example III were dissolved in 50 ml
of THF and cooled to 4.degree. C. with an ice bath. 156 mg (1.3 mmols) of
isoamyl nitrite and 130 mg (1.3 mols) of sulfuric acid were added, and
then 1.5 ml of water was added to dissolve the precipitate which formed.
After 2 hours, the reaction mixture was added dropwise to a stirring
solution of 100 mg (1.2 mmols) trifluorodimethyl sulfone, 2 ml 1N NaOH, 25
ml of 10% sodium acetate, and 25 ml water. After 1.5 hr, the reaction was
extracted with two 50 ml portions of ethyl ether. The organic phase was
dried with MgSO.sub.4 and concentrated in vacuo. The residue was
chromatographed on 3-20.times.20 cm 1000 micron Analtech silica gel GF
plates, eluant (2/8) hexane-ethyl acetate. The appropriate bands were
extracted with ethyl acetate and concentrated, yielding 210 mg of product
XI.
EXAMPLE VI
7-carbonitrile-2,12-dimethoxycarbonyl-4,10-dinitro-16,17-dihydro-5H,15H
dibenzo [b,i] [1,11,4,5,7,8] dioxatetraacyclotetradecin (XIII)
A 150-ml round-bottom flask equipped with a magnetic stir bar was charged
with 1.0 g (0.0028 mols) of product IX and 30 ml of glacial acetic acid.
To this stirring solution was added dropwise an ice-cooled solution of
0.152 ml concentrated H.sub.2 SO.sub.4 in 100 ml of glacial acetic acid
and the solution had a slight pink color and a precipitate was formed. 6
ml of 90% fuming nitric acid were added dropwise to an ice cold 20 ml
solution of glacial acetic acid and the resulting solution was added
dropwise to the above diamine solution. The reaction mixture was stirred
with heating at 55.degree. C. for 32 hours.
The reaction was poured into 200 ml CH.sub.2 Cl.sub.2 and extracted several
times with 200 ml ice water. The organic phase was extracted with four 200
ml portions of 0.1N NaOH, dried over MgSO.sub.4 and concentrated in vacuo.
Chromatography on 10-20.times.20 cm 1000 micron Analtech silica gel GF
plates, eluant (1/9) MeOH/CH.sub.2 Cl.sub.2. The appropriate bands were
extracted and subjected again to the above chromatography conditions. 0.97
g of 3,3'-(1,3-dioxopropyl)-4,4'-diamino-5,5'-dinitro bismethyl benzoate
(XII) were recovered, and had a melting point of 208.degree.-210.degree.
C.
100 g (0.215 mmols) of XII were dissolved in 25 ml THF and cooled to
4.degree. C. in an ice bath with stirring and then 51 mg (0.430 mmols) of
isoamyl nitrite and 57.2 .mu.l H.sub.2 SO.sub.4 were added and stirred for
3 hours. The resulting solution was added dropwise to 40 mg (0.470 mmols)
of cyanoacetic acid which had been dissolved in 5 ml 1N NaOH and 100 ml
H.sub.2 O at 4.degree. C. After 30 minutes, 80 mg of cyanoacetic acid were
added and allowed to stir for 3 hours at room temperature. The crude
reaction mixture was extracted with three 100 ml portions of CH.sub.2
Cl.sub.2, dried MgSO.sub.4, filtered and concentrated.
Purification by preparative TLC on four 20.times.20 cm, 1000 micron
Analtech silica gel GF, eluant ethyl acetate. The appropriate band was
extracted with methanol dichloromethane. The dry product was taken up in
dichloromethane and filtered through a fine glass funnel and 20 mg of
compound XIII were recovered.
EXAMPLE VII
Saponification of 3,3'-(1,3-dioxopropyl) 4,4'-dinitrobismethyl benzoate
(VIII)
8 g (0.022 mols) of product VIII was dissolved in 30 ml 1N NaOH, 150 ml
THF, and 40 ml MeOH. The solution was stirred at ambient temperature for
about 16 hr. To the clear reaction mixture 350 ml of water were added,
followed by dropwise addition of concentrated HCl. The white precipitate
was filtered and washed with 500 ml water. The resulting white solid was
dried over P.sub.2 O.sub.5 under vacuum to yield 9.3 g of
3,3'-(1,3-dioxopropyl-4,4'-bisbenzoic acid (XIV) with a melting point
greater than 300.degree. C.
EXAMPLE VIII
3,3'-(1,3-dioxopropyl)-4,4'-diaminobisbenzoic acid (XV)
A mixture of 3 grams of the product of Example VII (3 g) 200 ml water, and
sufficient concentrated NH.sub.4 OH to cause dissolution was hydrogenated
under 50 psi hydrogen pressure for 3 hours.
The solution was filtered and concentrated in vacuo. The resulting light
brown solid was dried over P.sub.2 O.sub.5 under vacuum to yield 2.5 g of
the product (XV), with melting point 234.degree.-235.degree. C.
EXAMPLE IX
7-trifluoromethylsulfonyl-2,12-dicarboxyl-16,17-dihydro-5H,15H, dibenzo
[b,i] [1,11,4,5,7,8] dioxatetraazacyclotetradecin (XVI)
500 mg (1.44 mmols) of the product of Example VIII were dissolved in 0.25M
HCl with heating, and 6 ml of THF were added, and the solution was stirred
and cooled in an ice bath to .sup..about. 5.degree. C. 200 mg (2.88 mols)
sodium nitrite dissolved in 10 ml H.sub.2 O were added dropwise to the
solution and it turned light green yellow. After 1 hour, the diazonium
solution was added dropwise to a vigorously stirring solution of 40 ml
0.25N NaOH, 20 ml THF, and 850 mg (5.76 mmols) of trifluorodimethyl
sulfone. The pH was increased during the reaction to 9.5-10 with 1N NaOH.
After 30 minutes, the product was acidified with concentrated HCl,
extracted twice with 200 ml ethyl ether, dried with MgSO.sub.4, filtered
and concentrated. Purification of 50 mg of crude product by column
chromatography using 5.times.16 cm column of Merck silanised silica gel 60
PF-254, eluant ethyl ether. Fractions were pooled, filtered through a
fine-glass funnel, and concentrated. The residue was dissolved in 10 ml
water with NH.sub.4 OH, precipitated with HCl and centrifuged. The pellet
was resuspended in water and centrifuged. The pellet was then dried at
100.degree. C. over P.sub.2 O.sub.5 under vacuum and 25.6 mg of XVI having
molecular weight 517.4 were recovered.
EXAMPLE X
7-nitro-2,12-dicarboxyl-16,17-dihydro-5H,15H-dibenzo [b,i] [1,11,4,5,7,8]
dioxatetraacyclotetradecin (XVII)
800 mg of the product of Example VIII, 150 ml of water, and 4 ml of
concentrated hydrochloric acid were mixed and then cooled to 5.degree. C.,
whereupon 637 mg (9.2 mmols) of sodium nitrite were added. After stirring
for 2.5 hours in an ice bath, 554 mg (9.2 mmols) of nitromethane were
added at once to the solution, and concentrated NH.sub.4 OH was added and
the resulting solution had pH 8.0. After 2.5 hours, the solution was made
acidic (pH 6.0) with concentrated HCl, and the precipitate was centrifuged
and the solid washed twice with 200 ml of water, centrifuged and dried at
100.degree. C. over P.sub.2 O.sub.5 in vacuo. The product contained
impurities that remain at base line using TLC silica gel GF, (0.5/95)
NH.sub.4 OH-MeOH system. To purify the product, an aliquot of the material
was suspended in a minimum amount of methanol and concentrated NH.sub.4 OH
was added to cause dissolution, then applied atop a dry packed column
4.times.15 cm of E. Merck silica gel 60 and eluted with 1% NH.sub.4
OH-MeOH. After monitoring by TLC silica gel GF, eluant 5 drops NH.sub.4
OH/10 ml MeOH, the appropriate fractions were pooled and concentrated in
vacuo. The residue was taken up in water NH.sub.4 OH and acidified with
HCl and centrifuged. This was repeated twice on the pellet and then washed
with methanol. The pellet was dried at 100.degree. C. over P.sub.2 O.sub.5
in vacuo, yielding 150 mg of compound XVII having a molecular weight of
430.4.
EXAMPLE XI
7-nitro-2,12,di(N-carbonyl glycine)-16,17-dihydro-5H,15H-dibenzo [b,i]
[1,11,4,5,7,8] dioxatetraacyclotetradecin (XXI)
2.5 g (0.0073 mols) of the product of Example VII, 1.5 g (0.0073 mols) of
N,N'-dicyclohexylcarbodiimide, 0.839 g (0.0073 mols) of NHS, and 50 ml of
anhydrous DMF were mixed and stirred at ambient temperature for 16 hours.
4.6 g (0.0366 mols) of glycine methyl ester hydrochloride were dissolved
in 40 ml DMF and 3.7 g (0.0366 mols) of triethylamine were added, and
after 1/2 hour, this solution was filtered rapidly into the above
activated NHS solution and left stirring overnight. The reaction mixture
was concentrated under high vacuum to a residue, and was taken up in 100
ml dichloromethane and filtered. The filtrate was extracted twice with 100
ml of 0.1N HCl dried MgSO.sub.4, filtered and concentrated.
Half of the residue was chromatographed using TLC on 20-20.times.20 cm 1000
micron Analtech silica GF plates and eluted with 5% MeOH-95% CH.sub.2
Cl.sub.2. The appropriate bands were pooled, extracted with MeOH-CH.sub.2
Cl.sub.2 and concentrated and yielded 1.6 g of product XVIII with
molecular weight 548.
1.5 grams of compound XVIII, 25 ml 1N NaOH, 100 ml THF, and 20 ml methanol
were mixed and stirred for 16 hr at room temperature. TLC analysis
Analtech silica gel GF (1/.9) MeOH-CH.sub.2 Cl.sub.2 showed no starting
material remaining.
100 ml of water were added and the organic solvents removed on a rotary
evaporator. The solution was made acidic with concentrated HCl (pH 3.0). A
white precipitate (compound XIX) was collected and was dried over P.sub.2
O.sub.5 in vacuo, and yielded 1.2 g. Compound XIX has a molecular weight
of 520.4.
A mixture of 1.2 g (0.002 mols) of Compound XIX, 100 ml absolute methanol,
and .sup..about. 0.4 ml of concentrated NH.sub.4 OH and 100 mg 10%
palladium on carbon was hydrogenated under 50 psi hydrogen pressure for
2.5 hours. The catalyst was removed by filtration, and the filtrate was
concentrated in vacuo, yielding 0.88 g of a light violet product (compound
XX) having a molecular weight of 460.4.
400 mg (0.87 mmols) of compound XX, 150 ml of water, and 5 ml concentrated
HCl were mixed and the solution was cooled to .sup..about. 5.degree. C.,
and 120 mg (1.74 mmols) of sodium nitrite in 10 ml of water were added
dropwise. A light yellow clear solution was formed. After 3.5 hours, 106
mg (1.74 mmols) of nitromethane were added and the solution stirred
vigorously with the dropwise addition of concentrated NH.sub.4 OH. A deep
red solution formed, and the pH was maintained below 8.5. After stirring
for 1.5 hours, the reaction was brought to pH 5.0 with hydrochloric acid.
The precipitate was centrifuged, and the pellet was resuspended in water
and centrifuged. This procedure was repeated using methanol. The pellet
was dried in vacuo over P.sub.2 O.sub.5 at 100.degree. C. The crude
product was dissolved in MeOH-NH.sub.4 OH, absorbed on Baker silica gel
60, and applied atop a 2.5.times.12 cm silica gel 60 column, eluted with
4% NH.sub.4 OH-96% MeOH and appropriate fractions pooled, concentrated in
vacuo. The residue was dissolved in NH.sub.4 OH-H.sub.2 O and precipitated
with HCl and centrifuged. The pellet was suspended in water and
centrifuged. The pellet was dried over P.sub.2 O.sub.5 at 100.degree. C.
in vacuo. 210 mg of compound XXI were recovered.
EXAMPLE XII
In order to demonstrate the efficacy of compounds of the present invention,
the compounds were employed in a number of assays for lithium. In carrying
out the assay a Gilford Stasar III spectrophotometer was employed, but any
spectrophotometer can be used. A pipettor-dilutor and a CP5000 printer
were also employed. The following solutions are prepared as reagents for
use in the assay.
Buffer:
55% DMSO/45% H.sub.2 O containing 0.055N KOH and 6.85 mM NaCl.
Complexing Agent:
9.3.times.10.sup.-4 M compound XVII in 55% DMSO/45% H.sub.2 O.
Calibrators:
1. Low: 1.25.times.10.sup.-4 M Li.sub.2 CO.sub.3 and 154 mM of NaCl in
H.sub.2 O corresponding to lithium ion concentration of 0.25 .mu.mole/ml
2. High: 7.5.times.10.sup.-4 M Li.sub.2 CO.sub.3 and 154 mM of NaCl in
H.sub.2 O corresponding to lithium ion concentration of 1.5 .mu.mole/ml
The protocol employed for carrying out an assay is as follows: 1) 60
microliters of the sample was drawn up and dispensed with 300 microliters
of buffer into a Croan cup. 2) 60 .mu.l of diluted serum was transferred
into a Croan cup and 300 .mu.l Buffer was then added. 3) 60 microliters of
the complexing agent were added to the diluted serum, followed by the
addition of 300 .mu.l of Buffer. The entire sample was mixed and aspirated
into the spectrophotometer. After 18 seconds a reading was taken. Emission
was measured at 550 nm.
A blank was carried out by repeating the above procedure using Buffer in
place of the Complexing Agent. Two to three readings were recorded for
both sample and blank. These values were averaged and then subtracted to
yield the final value for comparison with a standard curve.
The results are summarized below.
TABLE 1
______________________________________
Lithium ion Absorption
(.mu.mole/ml) (units)
______________________________________
0.25 2376
1.5 2570
______________________________________
For purposes of comparison, the correlation between the assay of the
present invention and atomic absorption assay was as follows:
Slope: 1.054
Intercept: -0.028
Corr: 0.994
S.E.E.: 0.03
N: 28 wherein S.E.E. is standard estimated error and N is the number of
patient samples.
The subject assay provides for a sensitive accurate method of determining
lithium in serum and the like. The subject invention provides novel
complexing agents for lithium which when employed in the method of the
present invention provide a method for qualitatively and quantitatively
determining lithium without deproteinization of the sample of interest,
such as serum. The method is rapid and the protocol is simple and
relatively free of technician introduced error. A two point standard curve
can be used for the assay. The assay can be carried out without the use of
enzyme and antibody reagents.
Although the foregoing invention has been described in some detail by way
of illustration and example for purposes of clarity and understanding, it
will be obvious that certain changes and modifications may be practiced
within the scope of the appended claims.
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